Nextera xt dna kit

Whole genome sequencing of selected samples was performed using a previously described MiSeq-based approach (Nguyen et al., 2016 (link)). Briefly, 110μl of the selected swab in viral transport medium was centrifuged at 13,000 rpm for 10 min to remove host cells or large cell debris. The collected supernatants were treated with DNase at 37°C for 30 min. Viral nucleic acid was then extracted using QIAamp viral RNA kit (Qiagen, Hilden, Germany) and recovered in 50 μL elution buffer provided with the kit. cDNA was synthesized from 10 ul of viral nucleic acid using Super Script III enzyme (Invitrogen, Carlsbad, CA, United States) and FR26RV-Endoh primer. This was followed by the conversion of cDNA into double-stranded (ds) DNA using exo-Klenow (Invitrogen), and random amplification of the resulting dsDNA using Platinum PCR Supermix (Invitrogen) and FR20RV primer. PCR products were then purified using AMPure (Beckman Coulter, Indianapolis, IN, United States) and processed to the library preparation step using Nextera XT DNA kit (Illumina, San Diego, CA, United States), following the manufacturer’s instructions. Finally, the prepared library was sequenced using MiSeq reagent kit v3 (600 cycles, Illumina), which produced read lengths up to 2 × 300 bp, in a MiSeq platform (Illumina) (Nguyen et al., 2016 (link)).

Inoculation of the ECIV isolate onto EPC cells in four 175 cm² flasks at a high multiplicity of infection provided third-passage material harvested after 21 days post-infection when CPE was extensive. Cell culture supernatant was clarified at 5520× g for 20 min at 4 °C. The pelleted virus was obtained by centrifugation of the clarified supernatant at 100,000× g for 1.25 h at 4 °C. The viral pellet was resuspended in 360 μL of animal tissue lysis (ATL) buffer prior to extraction of viral genomic DNA using a DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. A DNA library was generated using a Nextera XT DNA Kit, and sequencing was performed using a V3 chemistry 600 cycle Kit on a MiSeq sequencer (Illumina, Germantown, MD, USA). De novo assembly of the paired-end reads was performed in SPAdes 3.5.0 genome assembly algorithm [22 (link)]. The quality of the genome assembly was verified by mapping the reads back to the consensus sequence in Bowtie 2 2.1.0 [23 (link)] and visually inspecting the alignment in Tablet 1.14.10.20 [24 (link)].

Isolates were cultured overnight in screw-capped glass tubes (Pyrex, Iwaki Glass, Tokyo, Japan) filled with Todd-Hewitt broth (BD Biosciences, San Jose, CA, USA) and supplemented with 0.2% yeast extract (BD Biosciences) (THY) at 37°C in an ambient atmosphere. Bacterial cells were lysed with 10 units/mL mutanolysin (Sigma, St. Louis, MO, USA), 10 mg/mL lysozyme (Wako, Osaka, Japan), and 0.5 mg/mL achromopeptidase (Wako), and genomic DNA was extracted from overnight cell cultures using a Maxwell^® RSC instrument with a Maxwell^® RSC PureFood GMO and Authentication Kit (Promega Corporation, Madison, WI, USA). Paired-end libraries were generated from extracted DNA with a Nextera^® XT DNA kit (Illumina, San Diego, CA, USA). Libraries were sequenced with Illumina instruments (HiSeq X and Miseq) and paired-end sequence reads (150 bp and 301 bp, respectively) were obtained.

Single-cell capture was performed by C1 single-cell auto prep system (Fluidigm) following the manufacturer’s instructions^{17 (link)}. The microfluidics circuit used was the C1™ Single-Cell mRNA-seq IFC, 17–25 µm. All 96 chambers were inspected under an inverted phase contrast microscope; only chambers containing a non-damaged single cell were considered for downstream analysis. For the cell lysis and cDNA synthesis, we used the SMARTer Ultra Low RNA kit for Illumina Sequencing (version 2, Clontech) and a C1 Auto Prep System instrument (Fluidigm) with the original mRNA Seq Prep script provided by the manufacturer (1772×/1773×, Fluidigm). We assessed cDNA quality on 2100 Bioanalyzer (Agilent) with the high sensitivity DNA chips (Agilent) and quantified the cDNA using Qubit dsDNA BR assay kit (Invitrogen). Sequencing libraries were prepared with 0.3 ng of pre-amplified cDNA using Nextera XT DNA kit (Illumina) according to manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2000 machine as 100 bp reads single-end.

Single cells were index-sorted individually by FACS (BD Influx 5) into wells of a 96-well PCR plate containing lysis buffer. scRNA-seq was performed as previously described (Nestorowa et al., 2016 (link), Picelli et al., 2014 (link), Wilson et al., 2015 (link)) for a total of 1152 single cells. The Illumina Nextera XT DNA kit was used to prepare libraries. Pooled libraries were sequenced on the Illumina HiSeq 4000 (single-end 125bp reads). Samples from all cell lines were included in each sequencing lane, to control for technical lane effects. We did not detect a significant batch effect.